Protein biomarker candidates for a range of malignancies are being discovered at an escalating pace presenting the enormous challenge of identifying those with clinical utility. NIH/NCI has established serum banks with linked clinical outcomes that are vital resources for screening and validating new cancer biomarkers. However, the increasing demand for the limited serum samples contained in these banks has created a bottle-neck in biomarker validation. In this project, we will address the need for new technology that will make more efficient use of valuable serum banks. We propose to develop and test a platform with a 20-fold or greater increase in the number of biomarkers that can be assayed with a 100ul unit volume of serum. In Phase I, we will develop and evaluate a model multiplexed immunoassay for cancer related biomarkers, and in the process adapt and validate novel, proprietary substrate and imaging technologies for this application. Initially, we will qualify a candidate substrate with novel composition as a support for immunoassays by comparing its performance to commercial protein-array substrates. Next, we will develop an array format Cancer Antigen (CA) 125 immunoassay with performance comparable to the CA125 ELISA. The CA125 assay will then act as a platform with which to evaluate alternative fluorescence labels for their compatibility with the multiplexing technology. The label with the highest performance will be used for subsequent assay development. Finally, a multiplexed immunoassay for three cancer-relevant markers will be developed, optimized and evaluated using the prototype imager and a panel of patient and control serum samples. In Phase II, a full-scale development program is planned to integrate assay automation and processing instrumentation, and for evaluating the multiplexed immunoassay system as an efficient technology for screening serum banks to validate biomarkers for cancer diagnosis and management. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]